Abstract
The platelet integrin alpha IIb beta 3 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/1,2-dimyristoylphosphatidy lglycerol vesicles after removal of the detergent by adsorption to Bio-Beads. These vesicles were then used to generate planar lipid bilayers. The binding of fluorochrome labeled fibrinogen or the peptide ligand Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) was monitored by total internal reflection fluorescence microscopy and a solid phase binding assay. Analysis of the kinetics revealed fast reversible formation of a fibrinogen/integrin precomplex (KD = 50 nM) followed by formation of a stable irreversible complex. This transition was monitored by measuring the fraction of precomplex which could be dissociated by addition of excess Gly-Arg-Gly-Asp-Ser (GRGDS). For the peptide, the KD was 1200 nM, and the rates of association and dissociation were faster than the time resolution of the method. Similar KD values were found by inhibition of fibrinogen binding to alpha IIb beta 3 in the immobilized receptor assay. Since the binding of fibrinogen was irreversible, KD values were dependent on the time period between fibrinogen incubation and peptide addition. These and results by other authors point to the biological importance of the biphasic binding process of fibrinogen to its receptor on platelets.
Highlights
The platelet integrin aIIb83 binds to fibrinogen and tracellular domains (Hynes, 1992)
Integrin Can Be Reconstituted Correctly into Homogeneous Vesicles”cuIIb~3was isolated from human platelets by purifying Triton X-100-solubilized membrane proteins byion
Dialyzed vesicles were characterized by electron microscopy, and proincE&:zion teinllipid ratios were determined by protein and phosphate assays
Summary
From the concentration of labeled GRGDSPC peptide and its fluorescence intensity in theunbound state, thespecific fluorescence per fluorophore was determined From this value and the integrin surface concentration, the ratio of ligand to total receptor was calculated at maximum binding. We tried to incorporate this pure integrin into lipid vesicles using a previously described dialysis method with octyl-8-D-glucopyranoside as detergent (Pytela et al, 1985). DMPCIDMPG Vesicles form Bilayers on a Quartz Slide by Direct Fusion-Fluorescently labeled aIIbp with two FITC molecules/integrin was incorporated into DMPC/DMPG(50/ 50, mol/mol) vesicles. These were injected into the TIRFM cell for bilayer formation as monitored by space-restricted excitation by the evanescent field at the quartz-buffer interface (Fig. 2).
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